Humic acid induces G1 phase arrest and apoptosis in cultured vascular smooth muscle cells

Abstract

Humic acid (HA) in well water used by the inhabitants for drinking is one of the possible etiological factors for Blackfoot disease (BFD). In this study, the ability of HA to inhibit cell cycle progression and induce apoptosis in cultured smooth muscle cells (SMCs; A7r5) was investigated. Treatment of the SMCs at various HA concentrations (25-200 microg/mL) resulted in sequences of events marked by apoptosis, as shown by loss of cell viability, morphology change, and internucleosomal DNA fragmentation. HA-induced apoptotic cell death that is associated with loss of mitochondrial membrane potential (Delta Psi m), cytochrome c translocation, caspase-3, -8, and -9 activation, poly ADP-ribose polymerase (PARP) degradation, dysregulation of Bcl-2 and Bax, and upregulation of p53 and phospholyrated p53 (p-p53) in SMCs. Flow cytometry analysis demonstrated that HA blocked cell cycle progress in the G1 phase in SMCs. This blockade of cell cycle was associated with reduced amounts of cyclin D1, CDK4, cyclin E, CDK2, and hyperphosphorylated retinoblastoma protein (pRb) in a time-dependent manner. Apparent DNA strand breaks (DNA damage) were also detected in a dose-dependent manner using Single-cell gel electrophoresis assay (comet assay). Furthermore, HA induced dose-dependent elevation of reactive oxygen species (ROS) level in SMCs, and antioxidant vitamin C and Trolox effectively suppressed HA-induced DNA damage and dysregulation of Bcl-2/Bax. Our findings suggest that HA-induced DNA damage, cell cycle arrest, and apoptosis in SMCs may be an underlying mechanisms for the atherosclerosis and thrombosis observed in the BFD endemic region.

Ozone Reacts With Carbon Black to Produce a Fulvic Acid-Like Substance and Increase an Inflammatory Effect

“…51,54 The sequestration of iron, and an associated deficiency of cell metal after exposure to the standard fulvic acid, initiates pathways leading to injury and disease. 52,[55][56][57][58][59][60][61] Accordingly, while CB-Air did not change the release of pro-inflammatory mediators, CB-O 3 significantly increased IL-6 and IL-8 levels. Exposure to other xenobiotic agents with an equivalent capacity to complex metal cations induces a comparable inflammatory response in humans.…”

A Fulvic Acid-like Substance Participates in the Pro-inflammatory Effects of Cigarette Smoke and Wood Smoke Particles

“…Endogenous iron, essential for host function, can be complexed by a polyanionic particle surface . Comparable to other compounds with a capacity to appropriate cell iron, the response to the functional metal deficiency will include oxidative stress, cell signaling, transcription factor activation, and release of pro-inflammatory mediators. − This response can be observed following human exposure to PM which contains carbonaceous compounds. , The sequestration of iron and an associated deficiency of cell metal after exposure to the standard FA and FA-like substance in cigarette smoke and WSP can initiate pathways leading to injury and disease. − Exposure to other xenobiotic agents with an equivalent capacity to coordinate metal cations impacts a comparable inflammatory and fibrotic injury in humans. − The spectrum of lung disease following smoking and air pollution particle exposure is wide and includes inflammatory injuries; these are proposed to be associated with a functional deficiency of iron resulting after sequestration of host cell metal by the HULIS including FA . Exposures to both cigarette smoke and wood smoke also increase the risk for lung fibrosis. − These associations between (1) smoking and exposures to air pollution particles and wood smoke and (2) inflammatory and fibrotic lung injuries are comparable to other exposures to other xenobiotic agents with an equivalent capacity to coordinate metal cations.…”

Cigarette Smoke Particle-Induced Lung Injury and Iron Homeostasis

“…36 By impacting a functional iron deficiency in the host, CSP exposure initiates inflammation similar to other particles. [71][72][73][74][75][76][77][78][79][80][81][82][83][84] This response includes increased oxidative stress, cell signaling, transcription factor activation, and release of mediators. [83][84][85][86][87][88][89][90][91] Cell exposures to compounds and substances with a capacity to complex iron, including CSP and other particles, correlate with increased oxidant generation.…”